SMCHD1 mutation spectrum for facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS) reveals disease-specific localization of variants in the ATPase domain

Richard JLF Lemmers, PhD1*, Nienke van der Stoep, PhD2, Patrick J van der Vliet, BSc1, Steven A Moore, PhD 3, David San Leòn Granado, PhD 1, Katherine Johnson, PhD 4, Ana Topf, PhD 4, Volker Straub, PhD 4, Teresinha Evangelista, MD 5, Tahseen Mozaffar,MD6, Virginia Kimonis,MD6, Rita Selvatici, PhD 7, Alessandra Ferlini, MD, PhD 7, Nicol C Voermans, MD, PhD8, Baziel van Engelen, MD, PhD 8, Sabrina Sacconi, MD, PhD 9, Rabi Tawil, MD 10, Meindert H. Lamers, PhD 11, Silvère M van der Maarel, PhD 1*

1 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands 2 Laboratory for Diagnostic Genome Analysis, Leiden University Medical Center, Leiden, The Netherlands 3 University of Iowa, Iowa City, IA, USA 4 Institute of Genetic Medicine, University of Newcastle, UK 5 Unité de Morphologie Neuromusculaire, Institut de Myologie, Paris, France 6 University of California, Irvine, CA, USA 7 Unit of Medical Genetics, University of Ferrara, Ferrara Italy 8 Department of Neurology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands 9 Centre de référence des Maladies neuromusculaires, Nice University Hospital, Nice, France 10 Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA 11 Department of Cell and Chemical Biology, Leiden University Medical Center, The Netherlands

*corresponding author Richard JLF Lemmers, PhD Department of Human Genetics Leiden University Medical Center Leiden, The Netherlands 0031-71-5269481 Email: [email protected]

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Supplementary Materials and Methods Genetic analysis of D4Z4 repeats Determination of the D4Z4 repeat size and allelic background was performed by Southern blotting and hybridization with probes p13E-11, 4qA and 4qB as described previously. Hybridization conditions slightly vary between the different laboratories. In our laboratory hybridization with probe p13E-11 was performed in a buffer with 10% Dextran sulphate, 1M NaCl, 50 mM Tris-HCl, pH 7.5, 1% SDS and 250ug/ml Salmon sperm DNA at 65°C. 4qA and 4qB hybridizations were done in a phosphate buffer with 10% polyethylene glycol 6000.1 Methylation at D4Z4 was determined either by Southern blotting at the FseI site in D4Z4 or by bisulphite conversion and PCR at the DR1 site.2 For Southern blotting based methylation analysis with probe p13E-11 we used the hybridization buffer with 10% Dextran sulphate (see before). Calculations of repeat size corrected methylation compared to controls (delta1) and SMCHD1 pathogenic variant carriers (delta2) were done as described previously.3

SMCHD1 sequencing and variant prediction SMCHD1 variants were identified by Sanger sequencing or by whole exome or whole genome sequencing (WES/WGS) followed by confirmation using Sanger sequencing. All variants identified in FSHD2, BAMS and controls have been submitted to the Leiden Open Variation Database (LOVD, www.lovd.nl). The putative effects of the SMCHD1 variants were investigated through prediction algorithms using Alamut Visual v.2.4.2 (Interactive Biosoftware, https://www.interactive- biosoftware.com/alamut-visual/) or Variant Effect Predictor (VEP) in Ensemble (https://www.ensembl.org/info/docs/tools/vep/index.html). This includes SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/) and Align GVGD (Grantham Variation and Grantham Deviation, http://agvgd.hci.utah.edu/about.php). Splicing predictions were done in Alamut.

Statistical analysis The FseI methylation level, delta1 and delta2 methylation score (in Supplementary figure 2) were compared using the unpaired t-test in Graphpad Prism 7. For the comparisons shown in Supplementary table 4, we used a Pearson chi-square test with Yates’ continuity correction in R 3.3.2. For the visualization, the R packages ggplot2 and trackViewer from R/Bioconductor were used.4

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Supplementary Tables

identified new nr publication variants variants 1 Lemmers et al., 2012 (PMID: 23143600)8 15 15 2 Sacconi et al., 2013 (PMID: 24075187)13 3 3 3 Mitsuhashi et al., 2013 (PMID: 24128691)10 1 1 4 Winston et al., 2015 (PMID: 24755953)15 1 1 5 Lemmers et al., 2015 (PMID: 25256356)3 51 36 6 Larsen et al., 2015 (PMID: 25370034)7 11 11 7 Smith et al., 2015 (poster ASHG 2015) 8 8 8 Lemmers et al., 2015 (PMID: 25820463)9 2 2 9 Boogaard et al., 2016 (PMID: 25782668)14 5 5 10 Hamanaka et al., 2016 (PMID: 27061275)6 11 11 11 Gaillard et al., 2016 (PMID: 27634379)5 1 1 12 Nguyen et al., 2017 (PMID: 28744936)12 1 1 13 Mul et al., 2018 (PMID: 29980640)11 23 6 Total 101

Supplementary table 1 Chronological overview of 101 published SMCHD1 variants involved in FSHD2.3, 5-15 Third column shows all variants described in the publication. Some variants were described in consecutive publications and overlap. In the last column we only mention the non-overlapping variants.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 SMCHD1 SMCHD1 SMCHD1 EXAC (Minor Allele Frequency) Ensemble Alamut Total (1+2+3) ID Family Nr M/F cDNA (NM_015295.2) (hg19 (GRCh37.p5) (NP_056110.2) type ORF position FseI delta1 delta2 dbSNP AFR EUR EAS SAS PolyPhen (1) SIFT(2) AGVGD (3) GV GD Prediction Publication F1 Rf871 1 M c.101G>C g.2656175G>C R34P M P-ORF exon 1 11 -37 -3 probably_damaging(0.969) Deleterious(0) C0 241 40 2 F2 LOVD12 1 c.311A>G g.2666917A>G N104S M P-ORF exon 3 NA NA NA rs375795924 1,13E-04 7,99E-05 8,38E-04 0 benign(0) Tolerated(0.13) C0 223 0 0 ASHG conference 2015 (Smith, 2015) F3 Rf6 30 F c.320T>C g.2666926T>C L107P M P-ORF exon 3 3 -35 -6 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F4 LOVD11 1 M c.328G>A g.2666934G>A A110T M P-ORF exon 3 NA NA NA possibly_damaging(0.863) Deleterious(0.01) C0 27 37 2 PMID25370034 (Larsen, 2014) F5 Rf866 1 M c.410G>A g.2667016G>A G137E M P-ORF exon 3 22 -21 10 rs1057519644 probably_damaging(0.989) Deleterious(0.01) C0 79 30 2 PMID: 25256356 (Lemmers, HMG 2015) F6 LOVD21 1 c.448G>C g.2673303G>C D150H M P-ORF exon 4 NA NA NA probably_damaging(1) Deleterious(0) C65 0 81 3 ASHG conference 2015 (Smith, 2015) F7 Rf1685 1 M c.562G>A g.2674068G>A G188R M P-ORF exon 5 6 NA NA probably_damaging(1) Deleterious(0) C65 0 125 3 F8 LOVD23 1 c.565A>G g.2674071A>G M189V M P-ORF exon 5 NA NA NA probably_damaging(0.977) Deleterious(0) C15 0 21 2 ASHG conference 2015 (Smith, 2015) F9 Rf1142 1 M c.580C>T g.2674086C>T L194F M P-ORF exon 5 12 -28 2 probably_damaging(0.999) Deleterious(0) C15 0 22 2 PMID: 25256356 (Lemmers, HMG 2015) F10 Rf677 1 M c.610A>G g.2674116A>G K204E M P-ORF exon 5 6 -32 -4 rs1184311800 possibly_damaging(0.851) Deleterious(0) C55 0 57 3 PMID: 29980640 (Mul, 2018) F11 IW1 2 M c.724G>A g.2688477G>A A242T M P-ORF exon 6 14 NA NA probably_damaging(0.999) Deleterious(0) C55 0 58 3 PMID: 29980640 (Mul, 2018) F12 Rf1247 1 F c.787C>G g.2688659C>G H263D M P-ORF exon7 4 -34 -5 probably_damaging(0.998) Deleterious(0) C65 0 81 3 PMID: 25256356 (Lemmers, HMG 2015) F13 Rf2542 1 M c.790G>A g.2688662G>A E264K M P-ORF exon7 6 -33 -4 probably_damaging(0.996) Deleterious(0) C55 0 57 3 F14 LOVD36 1 M c.848A>G g.2688720A>G Y283C M P-ORF exon 7 4 NA NA rs886041921 probably_damaging(0.996) Deleterious(0) C55 22 192 3 PMID: 27061275 (Hamanaka, 2016) F15 Rf2484 2 F c.848A>G g.2688720A>G Y283C M P-ORF exon 7 5 -37 -6 rs886041921 probably_damaging(0.996) Deleterious(0) C55 22 192 3 F16 IW20 1 M c.1031G>A g.2694682G>A R344Q M P-ORF exon 9 24 NA NA rs370983669 4,11E-04 3,02E-05 0 0 benign(0.031) Deleterious(0) C35 0 43 2 PMID: 29980640 (Mul, 2018) F17 Rf742 1 M c.1058A>G g.2697047A>G Y353C M P-ORF exon 9 9 -41 -6 probably_damaging(0.997) Deleterious(0) C65 0 194 3 PMID: 23143600 (Lemmers, 2010) F18 Rf959 3 M c.1058A>G g.2697047A>G Y353C M P-ORF exon 9 12 NA NA probably_damaging(0.997) Deleterious(0) C65 0 194 3 PMID: 25256356 (Lemmers, HMG 2015) F19 Rf1196 1 F c.1273G>A g.2697970G>A G425R M P-ORF exon 10 1 -37 -8 probably_damaging(0.925) Deleterious(0) C65 0 125 3 PMID: 25256356 (Lemmers, HMG 2015) F20 IW35 1 M c.1273G>A g.2697970G>A G425R M P-ORF exon 10 17 NA NA probably_damaging(0.925) Deleterious(0) C65 0 125 3 F21 IW39 1 M c.1282C>T g.2697979C>T R428C M P-ORF exon 10 14 NA NA probably_damaging(0.996) deleterious(0) C65 0 179 3 F22 LOVD1 1 M c.1433G>A g.2700627G>A G478E M P-ORF exon 11 12 NA NA benign(0.142) Deleterious(0) C65 0 98 2 PMID25370034 (Larsen, 2014) F23 Rf739 1 M c.1436G>C g.2700630G>C R479P M P-ORF exon 11 10 -32 -1 probably_damaging(0.997) Deleterious(0) C65 0 103 3 PMID: 23143600 (Lemmers, 2010) F24 Rf2377 1 M c.1436G>T g.2700630G>T R479L M P-ORF exon 11 13 -31 1 probably_damaging(0.995) Deleterious(0) C65 0 102 3 PMID: 28744936 (Nguyen, 2017) F25 Rf2456 1 M c.1436G>A g.2700630G>A R479Q M P-ORF exon 11 2 -48 -13 probably_damaging(0.992) Deleterious(0) C35 0 43 3 F26 Rf300 1 M c.1474T>C g.2700743T>C C492R M P-ORF exon 12 7 -29 -2 probably_damaging(0.996) Deleterious(0) C65 0 180 3 PMID: 23143600 (Lemmers, 2010) F27 Rf1729 1 F c.1556T>C g.2700825T>C F519S M P-ORF exon 12 11 -31 -1 probably_damaging(0.994) Deleterious(0) C65 0 155 3 PMID: 29980640 (Mul, 2018) F28 Rf1021 1 M c.1580C>T g.2700849C>T T527M M P-ORF exon 12 18 -25 6 rs397518422 probably_damaging(0.992) Deleterious(0) C0 78 52 2 PMID: 24075187 (Sacconi, 2013) F29 IW31 1 M c.1652A>G g.2703694A>G Q551R M P-ORF exon 13 15 NA NA probably_damaging(0.968) Deleterious(0.03) C0 53 5 2 F30 Rf2572 1 M c.1786T>G g.2703828T>G W596G M P-ORF exon 13 10 -21 3 probably_damaging(0.994) Deleterious(0) C65 0 184 3 F31 LOVD5 1 F c.1844T>A g.2705693T>A V615D M P-ORF exon 14 17 NA NA probably_damaging(0.996) Deleterious(0) C65 0 152 3 PMID25370034 (Larsen, 2014) F32 IW8 1 F c.1865C>T g.2705714C>T P622L M P-ORF exon 14 8 NA NA probably_damaging(0.997) Deleterious(0) C65 0 98 3 F33 IW11 1 F c.1921G>T g.2705770G>T V641L M P-ORF exon 14 16 NA NA rs377559548 0 1,51E-05 0 0 benign(0.079) Deleterious(0.03) C0 29 5 1 F34 Rf399 3 F c.2068C>T g.2707565C>T P690S M P-ORF exon 16 7 -28 -1 rs397514623 probably_damaging(0.996) Deleterious(0) C65 0 73 3 PMID: 23143600 (Lemmers, 2010) F35 Rf917 1 M c.2243T>C g.2707901T>C L748P M P-ORF exon 17 11 -36 -3 possibly_damaging(0.905) Deleterious(0) C45 14 87 3 PMID: 25256356 (Lemmers, HMG 2015) F36 Rf1857 1 c.2321A>G g.2718216A>G Y774C M P-ORF exon 18 6 NA NA probably_damaging(0.921) Deleterious(0.01) C25 102 155 3 F37 Rf676 1 F c.2545G>A g.2722603G>A D849N M P-ORF exon 20 5 -37 -6 probably_damaging(0.996) Deleterious(0) C15 0 23 2 PMID: 23143600 (Lemmers, 2010) F38 Rf1432 1 F c.2768T>C g.2726517T>C L923P M P-ORF exon 22 5 NA NA probably_damaging(0.942) Deleterious(0) C0 112 53 2 F39 Rf1492 1 M c.2933T>A g.2729292T>A L978H M P-ORF exon 24 14 -28 3 probably_damaging(0.998) Deleterious(0) C0 235 89 2 F40 Rf385 101 M c.2941T>G g.2729300T>G Y981D M P-ORF exon 24 21 -24 8 benign(0.158) Deleterious(0) C35 56 140 2 PMID: 27153398 (Boogaard, 2016) F41 LOVD17 1 c.3187G>C g.2732401G>C G1063R M P-ORF exon 25 NA NA NA probably_damaging(0.999) Deleterious(0) C65 0 125 3 ASHG conference 2015 (Smith, 2015) F42 LOVD20 1 c.3323T>C g.2738441T>C L1108P M P-ORF exon 26 NA NA NA possibly_damaging(0.888) Deleterious(0.05) C15 106 80 2 ASHG conference 2015 (Smith, 2015) F43 IW3 1 M c.3340G>A g.2738458G>A V1114I M P-ORF exon 26 10 NA NA rs778206654 0 1,13E-04 0 5,80E-04 probably_damaging(0.956) Deleterious(0) C25 0 29 3 F44 Rf1659 1 F c.3811G>T g.2747529G>T V1271L M P-ORF exon 30 18 NA NA benign(0.087) Deleterious(0) C25 0 31 2 F45 Rf1430 1 F c.3899T>A g.2747617T>A I1300K M P-ORF exon 30 13 NA NA rs764012354 0 2,43E-05 0 0 probably_damaging(0.986) Deleterious(0) C35 29 91 3 F46 LOVD7 1 F c.4388A>C g.2760691A>C Q1463P M P-ORF exon 35 13 NA NA probably_damaging(0.969) Deleterious(0) C65 0 75 3 PMID25370034 (Larsen, 2014) F47 Rf1126 204 F c.4404G>A g.2760707G>A M1468I M P-ORF exon 35 13 -29 1 benign(0) Tolerated(0.64) C0 163 0 0 PMID: 25256356 (Lemmers, HMG 2015) F48 LOVD8 1 M c.4454C>T g.2762122C>T P1485L M P-ORF exon 36 18 NA NA probably_damaging(0.996) Deleterious(0) C15 99 94 2 PMID25370034 (Larsen, 2014) F49 Rf683 1 F c.4661T>C g.2763729T>C F1554S M P-ORF exon 37 11 -27 1 possibly_damaging(0.477) Deleterious(0) C65 0 155 3 PMID: 23143600 (Lemmers, 2010) F50 Rf1853 1 M c.5249A>G g.2775805A>G D1750G M P-ORF exon 42 5 -27 -2 probably_damaging(0.999) Deleterious(0) C65 0 94 3 F51 IW23 1 M c.5249A>T g.2775805A>T D1750V M P-ORF exon 42 12 NA NA probably_damaging(0.999) Deleterious(0) C65 0 152 3 F52 IW6 1 M c.5537A>G g.2778227A>G Y1846C M P-ORF exon 44 11 NA NA probably_damaging(0.998) Deleterious(0) C65 0 194 3 F53 Rf385 102 F c.5596C>G g.2784496C>G R1866G M P-ORF exon 45 10 -31 -1 probably_damaging(0.995) Deleterious(0) C65 0 125 3 PMID: 27153398 (Boogaard, 2016) F54 Rf1807 1 M c.5597G>A g.2784497G>A R1866Q M P-ORF exon 45 14 -28 3 rs886044586 probably_damaging(0.992) Deleterious(0) C35 0 43 3 F55 Rf975 201 F c.24del g.2656098del D D-ORF exon 1 15 -38 -1 PMID: 25256356 (Lemmers, HMG 2015) F56 IW10 1 F c.173_174insCT g.2656247_2656248insCT D D-ORF exon 1 19 NA NA F57 Rf743 1 M c.182_183dup g.2656256_2656257dup D D-ORF exon 1 14 -30 2 F58 Rf1472 1 M c.182_183dup g.2656256_2656257dup D D-ORF exon 1 15 NA NA F59 Rf1817 1 c.201_210delACCTGAAGAA g.2666169delACCTGAAGAA D D-ORF exon 2 6 NA NA F60 Rf1101 2 F c.582dupT g.2674088dup D D-ORF exon 5 16 -29 4 PMID: 25256356 (Lemmers, HMG 2015) F61 IW7 1 M c.687_690delTAGTinsG g.2688440_2688443_delinsG D P-ORF exon 6 5 NA NA F62 Rf1819 1 c.728_730delTCT g.2688481delTCT D P-ORF exon 6 3 NA NA PMID: 29980640 (Mul, 2018) F63 LOVD27 1 M c.823_825del g.2688695_2688697del D P-ORF exon 7 11 NA NA PMID: 24128691 (Mitsuhashi, 2013) F64 Rf2259 1 F c.975_976delAG g.2694626_2694627del D D-ORF exon 8 14 -31 1 F65 Rf2359 2 F c.1036_1039del g.2694687_2694690del D D-ORF exon 8 11 NA NA F66 LOVD30 1 F c.1205_1268del g.2697902_2697965del D D-ORF exon 10 11 NA NA PMID: 27061275 (Hamanaka, 2016) F67 Rf1033 1 F c.1302_1306delTGATA g.2697999_2698003del D D-ORF exon 10 24 -25 9 rs387907319 PMID: 23143600 (Lemmers, 2010) F68 Rf2282 1 M c.1490_1492delAGA g.2700759_2700761delAGA D P-ORF exon 12 8 -37 -5 rs886043345 F69 Rf393 203 M c.1608del g.2700875del D D-ORF exon 12 19 -31 4 PMID: 23143600 (Lemmers, 2010) F70 IW26 1 F c.1672_1673delGA g.2703714_2703715delGA D D-ORF exon 13 10 NA NA F71 Rf1637 2 M c.2088_2138del g.2707585_2707635del D D-ORF exon 16 10 -34 -2 F72 Rf1469 1 M c.2129dup g.2707626dup D D-ORF exon 16 5 NA NA F73 IW25 1 F c.2142dupT g.2707639dupT D D-ORF exon 16 6 NA NA F74 LOVD14 1 c.2335dupA g.2718230dup D D-ORF exon 18 NA NA NA ASHG conference 2015 (Smith, 2015) F75 Rf1666 1 F c.2468delC g.2722526delC D D-ORF exon 20 11 -34 -2 F76 Rf1823 1 c.2523_2525delTAA g.2722580delTAA D P-ORF exon 20 6 NA NA F77 Rf909 206 F c.2665dupA g.2724958dup D D-ORF exon 21 9 -23 2 PMID: 25256356 (Lemmers, HMG 2015) F78 LOVD37 1 F c.3073_3075del g.2732287_2732289del D P-ORF exon 25 16 NA NA PMID: 27061275 (Hamanaka, 2016) F79 IW32 1 M c.3197delT g.2732411delT D D-ORF exon 25 20 NA NA F80 Rf1199 1 M c.3238_3239delGA g.2732452_2732453del D D-ORF exon 25 9 NA NA rs886043182 PMID: 25256356 (Lemmers, HMG 2015) F81 Rf941 1 M c.3527_3529delinsTGC g.2740713_2740715delinsTGC D P-ORF exon 28 7 -29 -2 PMID: 25256356 (Lemmers, HMG 2015) F82 LOVD38 1 M c.4118_4132del g.2750458_2750472del D P-ORF exon 32 8 NA NA PMID: 27061275 (Hamanaka, 2016) F83 Rf753 1 M c.4457_4462dup g.2762125_2762130dup D P-ORF exon 36 10 -39 -4 PMID: 25256356 (Lemmers, HMG 2015) F84 GMC 1 c.4614_4615insTATAATA g.2763682_2763683insTATAATA D D-ORF exon 37 11 NA NA PMID: 27634379 (Gaillard, 2016) F85 Rf1121 1 M c.4738del g.2769710delA D D-ORF exon 38 9 -39 -5 PMID: 24075187 (Sacconi, 2013) F86 LOVD31 1 M c.5787_5790del g.2796014_2796017del D D-ORF exon 46 15 NA NA PMID: 27061275 (Hamanaka, 2016) F87 IW14 1 F 2,5 Mb deletion SMCHD1 2,5 Mb deletion SMCHD1 D D-ORF 14 NA NA F88 Rf898 2 F 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 16 -29 3 PMID: 25820463 (Lemmers, Hum Mut. 2015) F89 Rf929 1 M 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 14 -25 4 PMID: 25820463 (Lemmers, Hum Mut. 2015) F90 Rf922 1 M c.412C>T g.2667018C>T N D-ORF exon 3 7 -31 -2 PMID: 25256356 (Lemmers, HMG 2015) F91 IW15 1 M c.907G>T g.2694558G>T N D-ORF exon 8 5 NA NA F92 Rf391 3 M c.1030C>T g.2694681C>T N D-ORF exon 8 14 -34 0 rs886041918 PMID: 25256356 (Lemmers, HMG 2015) F93 IW29 1 M c.1186C>T g.2697883C>T N D-ORF exon 10 7 NA NA SMCHD1 SMCHD1 SMCHD1 EXAC (Minor Allele Frequency) Ensemble Alamut Total (1+2+3) ID Family Nr M/F cDNA (NM_015295.2) gene (hg19 (GRCh37.p5) protein (NP_056110.2) type ORF position FseI delta1 delta2 dbSNP AFR EUR EAS SAS PolyPhen (1) SIFT(2) AGVGD (3) GV GD Prediction Publication F94 IW33 1 M c.1186C>T g.2697883C>T N D-ORF exon 10 14 NA NA F95 IW34 1 M c.1186C>T g.2697883C>T N D-ORF exon 10 13 NA NA F96 LOVD24 1 c.1594G>T g.2700863G>T N D-ORF exon 12 NA NA NA rs780874216 ASHG conference 2015 (Smith, 2015) F97 Rf1640 1 F c.1654C>T g.2703696C>T N D-ORF exon 13 15 -31 2 rs1224850132 F98 Rf1575 1 M c.1738A>T g.2703780A>T N D-ORF exon 13 15 NA NA F99 LOVD28 1 M c.1819A>T g.2703861A>T N D-ORF exon 13 13 NA NA PMID: 27061275 (Hamanaka, 2016) F100 Rf562 1 F c.2191C>T g.2707849C>T N D-ORF exon 17 16 -26 5 PMID: 25256356 (Lemmers, HMG 2015) F101 Rf2255 1 F c.2377C>T g.2718351C>T N D-ORF exon 19 4 -23 -1 F102 Rf947 1 M c.2656C>T g.2724949C>T N D-ORF exon 21 9 -28 0 rs201632358 PMID: 27153398 (Boogaard, 2016) F103 IW28 1 M c.2656C>T g.2724949C>T N D-ORF exon 21 6 NA NA rs201632358 F104 Rf1416 1 M c.2785C>T g.2728466C>T N D-ORF exon 23 10 NA NA F105 Rf1473 1 M c.3010A>T g.2729369A>T N D-ORF exon 24 13 NA NA F106 Rf1468 1 F c.3469G>T g.2739473G>T N D-ORF exon 27 9 NA NA F107 Rf1727 2 F c.3631C>T g.2740817C>T N D-ORF exon 28 10 -34 -2 F108 Rf1552 203 M c.4267C>T g.2751377C>T N D-ORF exon 33 20 -24 8 F109 LOVD29 1 M c.4267C>T g.2751377C>T N D-ORF exon 33 8 NA NA PMID: 27061275 (Hamanaka, 2016) F110 LOVD6 1 F c.4988T>G g.2771552T>G N D-ORF exon 40 16 NA NA PMID25370034 (Larsen, 2014) F111 Rf2369 4 F c.5005C>T g.2771569C>T N D-ORF exon 40 4 -38 -7 rs1241705515 F112 Rf1837 1 c.5065G>T g.2772260G>T N D-ORF exon 41 21 NA NA rs1174155307 F113 Rf400 3 M c.5383C>T g.2777820C>T N D-ORF exon 43 22 -25 8 rs867694014 PMID: 25256356 (Lemmers, HMG 2015) F114 Rf1256 1 M c.5383C>T g.2777820C>T N D-ORF exon 43 14 -33 0 rs867694014 F115 Rf629 6 F c.5602C>T g.2784502C>T N D-ORF exon 45 12 -27 2 rs1229050345 PMID: 25256356 (Lemmers, HMG 2015) F116 Rf1754 1 F c.5866G>T g.2796093G>T N D-ORF exon 46 9 -36 -4 F117 Rf1657 1 M c.5866G>T g.2796093G>T N D-ORF exon 46 11 NA NA F118 LOVD32 1 M c.187-6G>A g.2666150G>A S3 D-ORF intron 1 21 NA NA PMID: 27061275 (Hamanaka, 2016) F119 Rf1818 1 c.263A>G g.2666869A>G D88G S3+M P-ORF exon 3 17 NA NA rs200521548 0 2,04E-04 0 1,02E-03 benign(0) Tolerated(0.06) C0 247 0 0 F120 Rf702 1 M c.754-2A>G g.2688624A>G S3 P-ORF intron 6 8 -43 -7 PMID: 25256356 (Lemmers, HMG 2015) F121 Rf744 1 M c.1843-15A>G g.2705677A>G S3 P-ORF intron 13 19 -27 6 F122 Rf644 1 F c.1843-1G>T g.2705691G>T S3 P-ORF intron 13 5 -32 -4 PMID: 25256356 (Lemmers, HMG 2015) F123 IW2 1 F c.1843-1G>A g.2705691G>A S3 P-ORF intron 13 6 NA NA rs886043146 F124 Rf549 3 M c.2147-1G>C g.2707804G>C S3 P-ORF intron 16 9 -44 -7 PMID: 25256356 (Lemmers, HMG 2015) F125 LOVD13 1 c.2147-1G>C g.2707804G>C S3 P-ORF intron 16 NA NA NA ASHG conference 2015 (Smith, 2015) F126 IW4 1 F c.2147-1G>C g.2707804G>C S3 P-ORF intron 16 <25 NA NA F127 Rf1749 1 M c.2914-5A>G g.2729268A>G S3 D-ORF intron 23 8 -33 -3 rs1215623273 F128 Rf394 213 M c.3426-2A>G g.2739428A>G S3 D-ORF intron 26 3 -36 -7 F129 Rf1352 1 M c.3634-19A>G g.2743740A>G S3 D-ORF intron 28 20 -24 8 PMID: 25256356 (Lemmers, HMG 2015) F130 Rf1831 1 c.3928-3T>G g.2750038T>G S3 D-ORF intron 31 7 NA NA F131 Rf1034 1 M c.4347-236A>G g.2760414A>G S3 D-ORF intron 34 10 -29 0 F132 Rf1507 1 F c.4720-2del g.2769690del S3 P-ORF intron 37 18 -28 4 F133 LOVD34 1 M c.5367-1G>C g.2777803G>C S3 D-ORF intron 42 23 NA NA PMID: 27061275 (Hamanaka, 2016) F134 Rf1043 1 M c.5720-2A>C g.2795945A>C S3 D-ORF intron 45 14 -26 3 PMID: 25256356 (Lemmers, HMG 2015) F135 Rf1614 1 M c.5720-1G>C g.2795946G>C S3 D-ORF intron 45 27 NA NA F136 Rf691 3 M c.186+1G>A g.2656261G>A S5 P-ORF intron 1 10 -35 -3 F137 Rf833 1 F c.424+1G>A g.2667031G>A S5 P-ORF intron 3 5 -38 -7 PMID: 25256356 (Lemmers, HMG 2015) F138 LOVD35 1 M c.638+5G>C g.2674149G>C S5 D-ORF intron 5 19 NA NA PMID: 27061275 (Hamanaka, 2016) F139 Rf394 204 F c.873+1G>A g.2688746G>A S5 P-ORF intron 7 4 -33 -5 PMID: 25256356 (Lemmers, HMG 2015) F140 LOVD25 1 M c.1040+1G>A g.2694692G>A S5 D-ORF intron 8 11 NA NA rs1245372794 PMID: 24755953 (Winston, 2014) F141 Rf1426 1 M c.1040+5G>A g.2694696G>A S5 D-ORF intron 8 13 NA NA F142 Rf1470 1 F c.1131+2_1131+5del g.2697122_2697125del S5 D-ORF intron 9 10 NA NA F143 LOVD10 1 M c.1463+5G>A g.2700662G>A S5 D-ORF intron 11 NA NA NA PMID25370034 (Larsen, 2014) F144 Rf696 1 M c.1647+3A>G g.2700919A>G S5 D-ORF intron12 12 -40 -4 PMID: 23143600 (Lemmers, 2010) F145 Rf849 1 M c.2146G>A g.2707643G>A G716S S5+M D-ORF intron 16 15 -23 5 probably_damaging(0.96) Deleterious(0) C55 0 55 3 PMID: 25256356 (Lemmers, HMG 2015) F146 Rf2747 1 c.2260+1G>T g.2707919G>T S5 P-ORF intron 17 5 NA NA F147 Rf1353 1 M c.2260+5G>A g.2707923G>A S5 P-ORF intron 17 4 -43 -10 PMID: 25256356 (Lemmers, HMG 2015) F148 Rf1124 1 F c.2338+1G>T g.2718234G>T S5 P-ORF intron 18 22 NA NA F149 Rf1451 1 M c.2338G>A g.2718233G>A E780K S5+M P-ORF intron 18 19 NA NA rs886044357 probably_damaging(0.978) Deleterious(0.04) C0 98 46 2 F150 Rf1002 1 M c.2338G>T g.2718233G>T S5+N D-ORF intron 18 15 -25 4 PMID: 25256356 (Lemmers, HMG 2015) F151 Rf1262 1 M c.2458+2T>A g.2718434T>A S5 P-ORF intron 19 6 NA NA F152 LOVD16 1 c.2458+5G>A g.2718437G>A S5 P-ORF intron 19 NA NA NA ASHG conference 2015 (Smith, 2015) F153 Rf268 3 M c.2603G>A g.2722661G>A S868N S5+M D-ORF intron 20 9 -24 2 benign(0.346) Deleterious(0.01) C0 58 39 1 PMID: 23143600 (Lemmers, 2010) F154 Rf947 1 M c.2700+1G>T g.2724994G>T S5 D-ORF intron 21 9 -28 0 PMID: 27153398 (Boogaard, 2016) F155 IW28 1 M c.2700+1G>T g.2724994G>T S5 D-ORF intron 21 6 NA NA F156 Rf1825 1 c.2913+1G>A g.2728595G>A S5 D-ORF intron 23 10 NA NA F157 Rf1504 1 M c.2913+1G>A g.2728595G>A S5 D-ORF intron 23 12 NA NA F158 Rf725 1 F c.3048+1G>T g.2729408G>T S5 P-ORF intron 24 8 -35 -4 PMID: 25256356 (Lemmers, HMG 2015) F159 Rf1110 1 M c.3048+2T>C g.2729409T>C S5 P-ORF intron 24 8 -36 -4 PMID: 24075187 (Sacconi, 2013) F160 Rf1828 1 c.3273_3276+1delTAAAG g.2732486delTAAAG S5 P-ORF intron 25 4 NA NA F161 LOVD3 1 F c.3274_3276+1del g.2732488_ 2732492del S5+N P-ORF intron 25 12 NA NA PMID25370034 (Larsen, 2014) F162 LOVD9 1 F c.3274_3276+1del g.2732488_ 2732492del S5+N P-ORF intron 25 NA NA NA PMID25370034 (Larsen, 2014) F163 LOVD33 1 M c.3274_3276+1del g.2732488_2732491del S5 P-ORF intron 25 4 NA NA PMID: 27061275 (Hamanaka, 2016) F164 Rf844 1 M c.3274_3276+1del g.2732488_2732491del S5 P-ORF intron 25 7 -36 -5 PMID: 23143600 (Lemmers, 2010) F165 Rf874 2 F c.3274_3276+1del g.2732488_2732491del S5 P-ORF intron 25 15 -42 -3 PMID: 23143600 (Lemmers, 2010) F166 LOVD18 1 c.3274_3276+1del g.2732488_2732491del S5 P-ORF intron 25 NA NA NA ASHG conference 2015 (Smith, 2015) F167 LOVD2 1 F c.3276_3276+4del g.2732487_ 2732492del S5+N P-ORF intron 25 6 NA NA PMID25370034 (Larsen, 2014) F168 Rf580 1 M c.3276_3276+4del g.2732490_2732494del S5+N P-ORF intron 25 11 -30 0 PMID: 25256356 (Lemmers, HMG 2015) F169 Rf936 1 F c.3276_3276+4del g.2732490_2732494del S5+N P-ORF intron 25 6 -35 -5 PMID: 25256356 (Lemmers, HMG 2015) F170 Rf1418 1 M c.3276_3276+4del g.2732490_2732494del S5+N P-ORF intron 25 8 NA NA F171 Rf1414 1 M c.3276_3276+4del g.2732490_2732494del S5 P-ORF intron 25 6 -37 -6 PMID: 27153398 (Boogaard, 2016) F172 Rf1865 1 M c.3276+1G>A g.2732491G>A S5 P-ORF intron 25 8 -30 -2 F173 Rf878 2 F c.3276+4_3276+7del g.2732494_2732497del S5 P-ORF intron 25 16 -35 1 PMID: 25256356 (Lemmers, HMG 2015) F174 Rf1309 1 M c.3276+4_3276+7del g.2732494_2732497del S5 P-ORF intron 25 11 -31 -1 PMID: 25256356 (Lemmers, HMG 2015) F175 LOVD19 1 c.3276+4_3276+7delAGTA g.2732494_2732497delAGTA S5 P-ORF intron 25 NA NA NA ASHG conference 2015 (Smith, 2015) F176 Rf1786 1 M c.3276+5G>A g.2732495G>A S5 P-ORF intron 25 10 NA NA F177 Rf1155 1 M c.3425G>C g.2738543G>C R1142T S5+M P-ORF intron 26 12 NA NA benign(0.307) Tolerated(0.14) C0 106 25 0 F178 Rf649 1 M c.3801+1G>A g.2743927G>A S5 P-ORF intron 29 16 -27 4 rs886042417 PMID: 23143600 (Lemmers, 2010) F179 Rf1203 1 F c.3801+1G>A g.2743927G>A S5 P-ORF intron 29 16 -25 5 rs886042417 PMID: 25256356 (Lemmers, HMG 2015) F180 Rf879 1 M c.3801+2T>C g.2743928T>C S5 P-ORF intron 29 10 -33 -2 rs778079305 0 1,85E-05 0 0 PMID: 25256356 (Lemmers, HMG 2015) F181 IW24 1 F c.4007+1G>A g.2750121G>A S5 D-ORF intron 32 16 NA NA F182 IW9 1 F c.4008-4_4008-2delTTAinsCT g.2750344_2750346delTTAinsCT S5 D-ORF intron 32 15 NA NA F183 LOVD4 1 F c.4346G>A g.2752550G>A R1449K S5+M P-ORF intron 34 15 NA NA probably_damaging(0.967) Deleterious(0) C25 0 26 3 PMID25370034 (Larsen, 2014) F184 Rf1254 1 M c.4346+1G>C g.2752551G>C S5 D-ORF intron 34 9 -29 -1 PMID: 25256356 (Lemmers, HMG 2015) F185 Rf392 201 M c.4566G>A g.2762234G>A S5 P-ORF intron 36 23 -27 8 PMID: 23143600 (Lemmers, 2010) F186 Rf1014 1 M c.4566G>A g.2762234G>A S5 P-ORF intron 36 17 NA NA PMID: 23143600 (Lemmers, 2010) F187 Rf2408 1 F c.5547+5G>A g.2778242G>A S5 D-ORF intron 44 5 -29 -3 SMCHD1 SMCHD1 SMCHD1 EXAC (Minor Allele Frequency) Ensemble Alamut Total (1+2+3) ID Family Nr M/F cDNA (NM_015295.2) gene (hg19 (GRCh37.p5) protein (NP_056110.2) type ORF position FseI delta1 delta2 dbSNP AFR EUR EAS SAS PolyPhen (1) SIFT(2) AGVGD (3) GV GD Prediction Publication F3 Rf6 265 F c.320T>C g.2666926T>C L107P M P-ORF exon 3 13 -38 -2 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F3 Rf6 52 F c.320T>C g.2666926T>C L107P M P-ORF exon 3 11 -34 -2 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F3 Rf6 262 M c.320T>C g.2666926T>C L107P M P-ORF exon 3 7 -36 -5 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F3 Rf6 37 F c.320T>C g.2666926T>C L107P M P-ORF exon 3 6 -35 -5 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F3 Rf6 53 M c.320T>C g.2666926T>C L107P M P-ORF exon 3 7 -39 -6 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F3 Rf6 51 F c.320T>C g.2666926T>C L107P M P-ORF exon 3 3 -42 -10 rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 29980640 (Mul, 2018) F12 IW1 1 F c.724G>A g.2688477G>A A242T M P-ORF exon 6 19 NA NA probably_damaging(0.999) Deleterious(0) C55 0 58 3 PMID: 29980640 (Mul, 2018) F18 Rf742 4 M c.1058A>G g.2697047A>G Y353C M P-ORF exon 9 9 -42 -6 probably_damaging(0.997) Deleterious(0) C65 0 194 3 PMID: 23143600 (Lemmers, 2010) F28 Rf1021 3 M c.1580C>T g.2700849C>T T527M M P-ORF exon 12 11 -27 1 rs397518422 probably_damaging(0.992) Deleterious(0) C0 78 52 2 PMID: 24075187 (Sacconi, 2013) F28 Rf1021 2 F c.1580C>T g.2700849C>T T527M M P-ORF exon 12 24 -25 10 rs397518422 probably_damaging(0.992) Deleterious(0) C0 78 52 2 PMID: 24075187 (Sacconi, 2013) F34 Rf399 2 F c.2068C>T g.2707565C>T P690S M P-ORF exon 16 10 -30 -1 rs397514623 probably_damaging(0.996) Deleterious(0) C65 0 73 3 PMID: 23143600 (Lemmers, 2010) F40 Rf385 203 M c.2941T>G and c.5596C>G g.2729300T>G and g.2784496C>G Y981D M P-ORF exon 24 5 -38 -7 benign(0.158) Deleterious(0) C35 56 140 2 PMID: 27153398 (Boogaard, 2016) F40 Rf385 206 F c.2941T>G and c.5596C>G g.2729300T>G and g.2784496C>G Y981D M P-ORF exon 24 1 -37 -9 benign(0.158) Deleterious(0) C35 56 140 2 PMID: 27153398 (Boogaard, 2016) F47 Rf1126 205 M c.4404G>A g.2760707G>A M1468I M P-ORF exon 35 12 -35 -1 benign(0) Tolerated(0.64) C0 163 0 0 PMID: 25256356 (Lemmers, HMG 2015) F47 Rf1126 208 F c.4404G>A g.2760707G>A M1468I M P-ORF exon 35 22 -34 5 benign(0) Tolerated(0.64) C0 163 0 0 PMID: 25256356 (Lemmers, HMG 2015) F55 Rf975 101 M c.24del g.2656098del D D-ORF exon 1 27 -26 11 PMID: 25256356 (Lemmers, HMG 2015) F55 Rf975 301 F c.24del g.2656098del D D-ORF exon 1 13 -38 -3 PMID: 25256356 (Lemmers, HMG 2015) F55 Rf975 302 F c.24del g.2656098del D D-ORF exon 1 13 -36 -1 PMID: 25256356 (Lemmers, HMG 2015) F57 Rf743 2 F c.182_183dup g.2656256_2656257dup D D-ORF exon 1 12 -34 -1 F57 Rf743 5 F c.182_183dup g.2656256_2656257dup D D-ORF exon 1 9 -31 -1 F57 Rf743 4 M c.182_183dup g.2656256_2656257dup D D-ORF exon 1 17 -26 5 F60 Rf1101 1 M c.582dupT g.2674088dup N D-ORF exon 5 11 -28 1 PMID: 25256356 (Lemmers, HMG 2015) F67 Rf1033 2 F c.1302_1306delTGATA g.2697999_2698003del D D-ORF exon 10 10 -22 3 rs387907319 F69 Rf393 101 M c.1608del g.2700875del D D-ORF exon 12 12 -38 -3 PMID: 23143600 (Lemmers, 2010) F69 Rf393 302 F c.1608del g.2700875del D D-ORF exon 12 19 -35 2 PMID: 23143600 (Lemmers, 2010) F69 Rf393 303 M c.1608del g.2700875del D D-ORF exon 12 19 -26 6 PMID: 23143600 (Lemmers, 2010) F69 Rf393 305 M c.1608del g.2700875del D D-ORF exon 12 21 -33 4 PMID: 23143600 (Lemmers, 2010) F69 Rf393 206 F c.1608del g.2700875del D D-ORF exon 12 11 -36 -3 PMID: 23143600 (Lemmers, 2010) F71 Rf1637 4 F c.2088_2138del g.2707585_2707635del D D-ORF exon 16 8 -30 -2 F71 Rf1637 5 F c.2088_2138del g.2707585_2707635del D D-ORF exon 16 10 -31 -1 F77 Rf909 202 F c.2665dupA g.2724958dup D D-ORF exon 21 7 -22 1 PMID: 25256356 (Lemmers, HMG 2015) F77 Rf909 102 F c.2665dupA g.2724958dup D D-ORF exon 21 5 -28 -3 PMID: 25256356 (Lemmers, HMG 2015) F88 Rf898 3 M 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 12 -26 2 PMID: 25820463 (Lemmers, Hum Mut. 2015) F88 Rf898 4 F 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 12 -31 0 PMID: 25820463 (Lemmers, Hum Mut. 2015) F88 Rf898 1 M 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 13 -28 2 PMID: 25820463 (Lemmers, Hum Mut. 2015) F88 Rf898 5 M 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 16 -25 5 PMID: 25820463 (Lemmers, Hum Mut. 2015) F89 Rf929 3 F 1,2 Mb deletion SMCHD1 1,2 Mb deletion SMCHD1 D D-ORF 20 -25 7 PMID: 25820463 (Lemmers, Hum Mut. 2015) F90 Rf922 2 F c.412C>T g.2667018C>T N D-ORF exon 3 12 -42 -5 PMID: 25256356 (Lemmers, HMG 2015) F99 LOVD28 2 F c.1819A>T g.2703861A>T N D-ORF exon 13 10 NA NA PMID: 27061275 (Hamanaka, 2016) F102 Rf947 3 M c.2656C>T g.2724949C>T N D-ORF exon 21 8 -33 -3 rs201632358 F102 Rf947 2 F c.2656C>T g.2724949C>T N D-ORF exon 21 13 -26 3 rs201632358 F107 Rf1727 1 F c.3631C>T g.2740817C>T N D-ORF exon 28 13 -31 1 F108 Rf1552 201 M c.4267C>T g.2751377C>T N D-ORF exon 33 17 -27 5 F108 Rf1552 102 F c.4267C>T g.2751377C>T N D-ORF exon 33 17 -25 6 F113 Rf400 4 M c.5383C>T g.2777820C>T N D-ORF exon 43 18 -33 3 rs867694014 F115 Rf629 2 F c.5602C>T g.2784502C>T N D-ORF exon 45 8 -24 1 rs1229050345 PMID: 25256356 (Lemmers, HMG 2015) F115 Rf629 3 M c.5602C>T g.2784502C>T N D-ORF exon 45 3 -26 -3 rs1229050345 PMID: 25256356 (Lemmers, HMG 2015) F115 Rf629 4 M c.5602C>T g.2784502C>T N D-ORF exon 45 8 -21 2 rs1229050345 PMID: 25256356 (Lemmers, HMG 2015) F115 Rf629 5 M c.5602C>T g.2784502C>T N D-ORF exon 45 8 NA NA rs1229050345 F115 Rf629 10 F c.5602C>T g.2784502C>T N D-ORF exon 45 14 -26 3 rs1229050345 F115 Rf629 11 F c.5602C>T g.2784502C>T N D-ORF exon 45 12 -28 1 rs1229050345 F115 Rf629 12 F c.5602C>T g.2784502C>T N D-ORF exon 45 6 -29 -2 rs1229050345 PMID: 25256356 (Lemmers, HMG 2015) F118 LOVD32 2 M c.187-6G>A g.2666150G>A S3 D-ORF intron 1 10 NA NA PMID: 27061275 (Hamanaka, 2016) F121 Rf744 4 F c.1843-15A>G g.2705677A>G S3 P-ORF intron 13 10 -34 -3 F124 Rf549 1 M c.2147-1G>C g.2707804G>C S3 P-ORF intron 16 7 NA NA F129 Rf1352 2 M c.3634-19A>G g.2743740A>G S3 D-ORF intron 28 4 -28 -3 PMID: 25256356 (Lemmers, HMG 2015) F131 Rf1034 4 F c.4347-236A>G g.2760414A>G S3 D-ORF intron 34 9 -30 -1 F131 Rf1034 5 F c.4347-236A>G g.2760414A>G S3 D-ORF intron 34 9 -30 -1 F131 Rf1034 3 F c.4347-236A>G g.2760414A>G S3 D-ORF intron 34 29 -26 12 F136 Rf691 1 M c.186+1G>A g.2656261G>A S5 P-ORF intron 1 14 -31 1 F136 Rf691 5 M c.186+1G>A g.2656261G>A S5 P-ORF intron 1 9 -34 -3 F139 Rf394 301 F c.873+1G>A g.2688746G>A S5 P-ORF intron 7 10 -37 -4 F154 Rf947 3 M c.2700+1G>T g.2724994G>T S5 D-ORF intron 21 8 -33 -3 F154 Rf947 2 F c.2700+1G>T g.2724994G>T S5 D-ORF intron 21 13 -26 3 F159 Rf1110 3 F c.3048+2T>C g.2729409T>C S5 P-ORF intron 24 10 -41 -6 PMID: 24075187 (Sacconi, 2013) F165 Rf874 3 F c.3274_3276+1del g.2732488_2732491del S5 P-ORF intron 25 12 -35 -2 PMID: 23143600 (Lemmers, 2010) F171 Rf1414 4 F c.3276_3276+4del g.2732490_2732494del S5 P-ORF intron 25 6 -36 -5 F178 Rf649 3 F c.3801+1G>A g.2743927G>A S5 P-ORF intron 29 9 -30 -1 rs886042417 PMID: 23143600 (Lemmers, 2010) F178 Rf649 5 M c.3801+1G>A g.2743927G>A S5 P-ORF intron 29 13 -31 1 rs886042417 PMID: 23143600 (Lemmers, 2010) F181 IW24 2 F c.4007+1G>A g.2750121G>A S5 D-ORF intron 32 13 NA NA F182 IW9 2 F c.4008-4_4008-2delTTAinsCT g.2750344_2750346delTTAinsCT S5 D-ORF intron 32 17 NA NA F184 Rf1254 3 F c.4346+1G>C g.2752551G>C S5 D-ORF intron 34 9 NA NA F184 Rf1254 2 F c.4346+1G>C g.2752551G>C S5 D-ORF intron 34 12 NA NA F185 Rf392 202 F c.4566G>A g.2762234G>A T1522T S5 P-ORF exon 36 11 -39 -4 PMID: 23143600 (Lemmers, 2010) F185 Rf392 102 F c.4566G>A g.2762234G>A T1522T S5 P-ORF exon 36 17 -30 3 PMID: 23143600 (Lemmers, 2010) F186 Rf1014 3 M c.4566G>A g.2762234G>A T1522T S5 P-ORF exon 36 16 NA NA PMID: 23143600 (Lemmers, 2010) F186 Rf1014 4 F c.4566G>A g.2762234G>A T1522T S5 P-ORF exon 36 24 -32 6 PMID: 23143600 (Lemmers, 2010) B1 NG15 1 F c.320T>C g.2666926T>C L107P M P-ORF exon 3 NA NA NA rs1135402737 probably_damaging(0.998) Deleterious(0) C65 0 98 3 PMID: 28067909 (Shaw, 2017) B2 Rf2324 1 M c.386T>A g.2666992T>A M129K M P-ORF exon 3 8 -40 -6 rs1135402738 benign(0.295) Deleterious(0.01) C0 81 46 1 PMID: 28067909 (Shaw, 2017) B3 IW27 1 F c.386T>G g.2666992T>G M129R M P-ORF exon 3 4 -40 -8 benign(0.369) Deleterious(0.01) C0 81 36 1 B4 NG11 1 F c.400G>T g.2667006G>T A134S M P-ORF exon 3 NA NA NA probably_damaging(0.991) Deleterious(0) C0 208 18 2 PMID: 2806791 (Gordon, 2017) B5 NG12 1 F c.400G>T g.2667006G>T A134S M P-ORF exon 3 NA NA NA probably_damaging(0.991) Deleterious(0) C0 208 18 2 PMID: 2806791 (Gordon, 2017) B6 NG2 1 F c.403A>T g.2667009A>T S135C M P-ORF exon 3 NA NA NA rs1057519645 probably_damaging(0.996) Deleterious(0) C65 0 112 3 PMID: 28067909 (Shaw, 2017); PMID: 2806791 (Gordon, 2017) B7 NG4 1 F c.403A>T g.2667009A>T S135C M P-ORF exon 3 15 NA NA rs1057519645 probably_damaging(0.996) Deleterious(0) C65 0 112 3 PMID: 28067909 (Shaw, 2017); PMID: 2806791 (Gordon, 2017) B8 IW18 1 M c.404G>A g.2667010G>A S135N M P-ORF exon 3 6 -40 -7 rs1057519646 probably_damaging(0.986) Deleterious(0) C45 0 46 3 PMID: 28067909 (Shaw, 2017) B9 NG16 1 F c.404G>A g.2667010G>A S135N M P-ORF exon 3 NA NA NA rs1057519646 probably_damaging(0.986) Deleterious(0) C45 0 46 3 PMID: 28067909 (Shaw, 2017) B10 NG3 1 M c.404G>A g.2667010G>A S135N M P-ORF exon 3 NA NA NA rs1057519646 probably_damaging(0.986) Deleterious(0) C45 0 46 3 PMID: 2806791 (Gordon, 2017) B11 NG17 1 M c.404G>T g.2667010G>T S135I M P-ORF exon 3 NA NA NA rs1057519646 probably_damaging(0.994) Deleterious(0) C65 0 142 3 PMID: 28067909 (Shaw, 2017) B12 NG1 1 M c.407A>G g.2667013A>G E136G M P-ORF exon 3 NA NA NA probably_damaging(0.994) Deleterious(0) C65 0 98 3 PMID: 2806791 (Gordon, 2017) B13 NG18 1 M c.408A>C g.2667014A>C E136D M P-ORF exon 3 18 -25 6 rs1057519643 probably_damaging(0.978) Deleterious(0) C35 0 45 3 PMID: 28067909 (Shaw, 2017) B14 Rf2320 1 F c.410G>A g.2667016G>A G137E M P-ORF exon 3 24 -23 10 rs1057519644 probably_damaging(0.989) Deleterious(0.01) C0 79 30 2 PMID: 28067909 (Shaw, 2017) B15 Rf2316 1 F c.415A>C g.2667021A>C N139H M P-ORF exon 3 16 -29 3 rs1135402739 probably_damaging(0.992) Deleterious(0.02) C0 94 15 2 PMID: 28067909 (Shaw, 2017) B16 Rf2328 1 F c.415A>C g.2667021A>C N139H M P-ORF exon 3 9 -34 -3 rs1135402739 probably_damaging(0.992) Deleterious(0.02) C0 94 15 2 PMID: 28067909 (Shaw, 2017) B17 Rf2323 1 M c.423G>C g.2667029G>C L141F M P-ORF exon 3 3 -39 -8 rs1057519641 probably_damaging(0.999) Deleterious(0) C15 0 22 2 PMID: 28067909 (Shaw, 2017) SMCHD1 SMCHD1 SMCHD1 EXAC (Minor Allele Frequency) Ensemble Alamut Total (1+2+3) ID Family Nr M/F cDNA (NM_015295.2) gene (hg19 (GRCh37.p5) protein (NP_056110.2) type ORF position FseI delta1 delta2 dbSNP AFR EUR EAS SAS PolyPhen (1) SIFT(2) AGVGD (3) GV GD Prediction Publication B18 NG19 1 F c.423G>C g.2667029G>C L141F M P-ORF exon 3 NA NA NA rs1057519641 probably_damaging(0.999) Deleterious(0) C15 0 22 2 PMID: 28067909 (Shaw, 2017) B19 Rf2325 1 M c.423G>C g.2667029G>C L141F M P-ORF exon 3 3 -38 -8 rs1057519641 probably_damaging(0.999) Deleterious(0) C15 0 22 2 PMID: 28067909 (Shaw, 2017) B20 NG20 1 M c.423G>C g.2667029G>C L141F M P-ORF exon 3 NA NA NA rs1057519641 probably_damaging(0.999) Deleterious(0) C15 0 22 2 PMID: 28067909 (Shaw, 2017) B21 NG21 1 M c.511T>G g.2674017T>G F171V M P-ORF exon 5 NA NA NA rs1135402740 possibly_damaging(0.795) Deleterious(0) C45 0 49 3 PMID: 28067909 (Shaw, 2017) B22 Rf2317 1 M c.725C>G g.2688478C>G A242G M P-ORF exon 6 NA NA NA rs1135402741 probably_damaging(0.998) Deleterious(0) C55 0 60 3 PMID: 28067909 (Shaw, 2017) B23 NG10 1 F c.1025G>C g.2694676G>C W342S M P-ORF exon 8 NA NA NA probably_damaging(0.996) Deleterious(0) C65 0 177 3 PMID: 2806791 (Gordon, 2017) B24 NG21 1 F c.1034A>G g.2694685A>G Q345R M P-ORF exon 8 NA NA NA rs1057519639 possibly_damaging(0.787) Deleterious(0.01) C0 29 43 2 PMID: 28067909 (Shaw, 2017) B25 Rf2326 1 M c.1043A>G g.2697032A>G H348R M P-ORF exon 9 NA NA NA rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017) B26 NG13 1 F c.1043A>G g.2697032A>G H348R M P-ORF exon 9 NA NA NA rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017); PMID: 2806791 (Gordon, 2017) B27 NG5 1 M c.1043A>G g.2697032A>G H348R M P-ORF exon 9 NA NA NA rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017); PMID: 2806791 (Gordon, 2017) B28 NG22 1 F c.1043A>G g.2697032A>G H348R M P-ORF exon 9 NA NA NA rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017) B29 NG23 1 M c.1043A>G g.2697032A>G H348R M P-ORF exon 9 NA NA NA rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017) B30 Rf2318 1 M c.1043A>G g.2697032A>G H348R M P-ORF exon 9 9 -28 0 rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017) B31 Rf2319 1 M c.1043A>G g.2697032A>G H348R M P-ORF exon 9 17 -29 4 rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 28067909 (Shaw, 2017) B32 NG14 1 F c.1043A>G g.2697032A>G H348R M P-ORF exon 9 NA NA NA rs1057519640 probably_damaging(0.955) Deleterious(0) C25 0 29 3 PMID: 2806791 (Gordon, 2017) B33 Rf2321 1 F c.1199A>T g.2697896A>T Q400L M P-ORF exon 10 8 -44 -8 rs1057519642 possibly_damaging(0.702) Deleterious(0) C65 0 112 3 PMID: 28067909 (Shaw, 2017) B34 NG6 1 M c.1259A>T g.2697956A>T D420V M P-ORF exon 10 NA NA NA rs1135402742 benign(0.296) Deleterious(0) C35 45 121 2 PMID: 28067909 (Shaw, 2017); PMID: 2806791 (Gordon, 2017) B35 NG9 1 M c.1259A>T g.2697956A>T D420V M P-ORF exon 10 NA NA NA rs1135402742 benign(0.296) Deleterious(0) C35 45 121 2 PMID: 2806791 (Gordon, 2017) B36 Rf2327 1 M c.1417G>C g.2700611G>C E473Q M P-ORF exon 11 NA NA NA rs1135402743 probably_damaging(0.981) Deleterious(0.01) C0 45 29 2 PMID: 28067909 (Shaw, 2017) B37 NG8 1 F c.1552A>G g.2700821A>G K518E M P-ORF exon 12 NA NA NA possibly_damaging(0.696) Deleterious(0.01) C15 26 44 2 PMID: 2806791 (Gordon, 2017) B38 NG24 1 M c.1568C>A g.2700837C>A T523K M P-ORF exon 12 NA NA NA rs1135402744 probably_damaging(0.994) Deleterious(0) C65 0 78 3 PMID: 28067909 (Shaw, 2017) B39 NG25 1 F c.1568C>A g.2700837C>A T523K M P-ORF exon 12 NA NA NA rs1135402744 probably_damaging(0.994) Deleterious(0) C65 0 78 3 PMID: 28067909 (Shaw, 2017) B40 Rf2322 1 M c.1571A>G g.2700840A>G N524S M P-ORF exon 12 NA NA NA rs1135402745 probably_damaging(0.985) Deleterious(0) C45 0 46 3 PMID: 28067909 (Shaw, 2017) B41 NG7 1 M c.1655G>A g.2703697G>A R552Q M P-ORF exon 13 NA NA NA rs886042392 probably_damaging(0.992) Deleterious(0) C35 0 43 3 PMID: 28067909 (Shaw, 2017); PMID: 2806791 (Gordon, 2017) B24 NG21 2 F c.1034A>G g.2694685A>G Q345R M P-ORF exon 8 NA NA NA rs1057519639 possibly_damaging(0.787) Deleterious(0.01) C0 29 43 2 PMID: 28067909 (Shaw, 2017) B33 Rf2321 3 M c.1199A>T g.2697896A>T Q400L M P-ORF exon 10 NA NA NA rs1057519642 possibly_damaging(0.702) Deleterious(0) C65 0 112 3 PMID: 28067909 (Shaw, 2017) B33 Rf2321 5 F c.1199A>T g.2697896A>T Q400L M P-ORF exon 10 NA NA NA rs1057519642 possibly_damaging(0.702) Deleterious(0) C65 0 112 3 PMID: 28067909 (Shaw, 2017) C1 Rf2071 1 c.116A>C g.2656190A>C E39A M P-ORF exon 1 53 NA NA rs990903424 benign(0) Deleterious(0.02) C0 227 39 1 C2 Rf1820 1 c.1186C>A g.2697883C>A Q396K M P-ORF exon 10 41 NA NA rs377471712 0 3,00E-05 0 0 benign(0) Tolerated(0.7) C0 139 0 0 C3 13 1 c.2122G>A g.2707619G>A V708I M P-ORF exon 16 Normal Normal NA rs2276092 0,719 0,702 0,610 0,638 benign(0) Tolerated(1) C0 89 0 0 C4 16 1 c.2637A>T g.2724930A>T K879N M P-ORF exon 21 Normal Normal NA rs633422 0,110 0,506 0,25 0,239 benign(0) Tolerated(0.1) C0 143 0 0 C5 Rf2541 1 M c.2837C>T g.2728518C>T A946V M P-ORF exon 23 54 5 40 possibly_damaging(0.629) Deleterious(0.04) C0 99 63 2 C6 Rf1827 1 c.3007A>G g.2729366A>G I1003V M P-ORF exon 24 43 NA NA rs777768323 0 8,23E-05 0 0 benign(0.158) Tolerated(0.06) C0 89 0 0 C7 Rf2372 2 c.3247A>G g.2732461A>G I1083V M P-ORF exon 25 Normal NA NA rs199901577 0 8,04E-05 0 0 benign(0.049) Deleterious(0.04) C15 5 29 1 C8 Rf1414 2 F c.3538G>A g.2740724G>A G1180R M P-ORF exon 28 62 18 50 rs768096462 0 1,50E-05 0 0 probably_damaging(0.999) Deleterious(0.01) C0 94 71 2 C9 Rf2520 1 c.3769C>T g.2743894C>T L1257F M P-ORF exon 29 47 NA NA rs766226383 0 1,64E-05 0 0 benign(0.228) Deleterious(0.01) C0 31 21 1 C10 Rf1795 1 c.3838C>G g.2747556C>G P1280A M P-ORF exon 30 50 NA NA rs1322896954 probably_damaging(0.994) Deleterious(0) C25 0 27 3 C11 Rf1229 4 F c.3872A>G g.2747590A>G N1291S M P-ORF exon 30 43 -2 31 rs201497685 0 4,54E-04 0 0 benign(0.357) Deleterious(0.04) C0 158 25 1 C12 Rf2278 1 M c.4226G>T g.2751336G>T R1409H M P-ORF exon 33 37 NA NA rs746741499 0 7,54E-05 0 0 benign(0.32) Deleterious(0.02) C15 99 98 1 C13 Rf1832 1 c.4328A>G g.2752532A>G D1443G M P-ORF exon 33 32 NA NA rs756531175 0 1,61E-05 0 0 benign(0.043) Deleterious(0.02) C0 260 0 1 C14 Rf1833 1 c.4544G>A g.2762212G>A R1515K M P-ORF exon 36 58 NA NA rs372451234 0 4,50E-05 0 0 benign(0) Tolerated(1) C0 65 0 0 C15 Rf1846 1 M c.4599G>T g.2763667G>T L1533F M P-ORF exon 37 Normal Normal NA rs770018521 0 5,14E-05 0 0 possibly_damaging(0.462) Deleterious(0.01) C0 14 21 2 C16 Rf1836 1 c.4786C>T g.2769758C>T R1596W M P-ORF exon 38 47 NA NA rs746646525 0 3,14E-05 0 1,85E-04 benign(0.109) Tolerated(0.05) C0 188 47 0 C17 Rf2279 1 c.4849G>A g.2769989G>A V1617I M P-ORF exon 39 38 NA NA rs774306466 0 1,54E-05 0 0 benign(0.026) Tolerated(0.1) C0 49 0 0 C18 Rf2430 1 F c.5317G>C g.2775873G>C V1773L M P-ORF exon 42 48 NA NA probably_damaging(0.994) Deleterious(0) C25 0 31 3 C19 Rf1567 1 c.5914C>T g.2796440C>T R1972C M P-ORF exon 47 64 NA NA rs778853203 0 0 0 1,04E-04 benign(0) Tolerated(0.08) C0 265 0 0 C20 Rf1405 1 M c.874-6T>C g.2694519T>C S3 P-ORF intron 7 25 -9 17 C21 Rf2430 1 F c.1132-14C>T g.2697815C>T S3 D-ORF intron 9 Normal NA NA rs184510956 2,14E-04 6,13E-04 0 0 C22 Rf1858 1 F c.1648-31A>G+c.1419A>G g.2703659A>G; g.2700613A>G S3 D-ORF intron12 35 -2 26 rs76103139 4,25E-04 3,95E-03 0 2,33E-03 C23 11 1 c.1843-17T>A g.2705675T>A S3 P-ORF intron 13 Normal Normal NA rs8090988 C24 Rf1449a 2 M c.1843-16A>T g.2705676A>T S3 P-ORF intron 13 59 16 47 rs569911568 1,24E-04 5,09E-04 0 2,14E-04 C25 Rf1220 4 F c.1957-3C>T g.2706359C>T S3 P-ORF intron 14 44 1 32 rs201069970 3,48E-04 1,07E-03 8,70E-04 3,99E-03 C26 15 1 c.2338+32A>G g.2718265A>G S3 P-ORF intron 17 Normal Normal NA rs62084229 0,216 0,119 0,0719 0,273 C27 21 1 c.3277-42_3277-39dupAGAC g.2738352T>TAGAC S3 P-ORF intron 25 Normal Normal NA rs71365196 0,273 0,297 0,510 0,448 C28 Rf1785 1 M c.4967-15G>T g.2771516G>T S3 P-ORF intron 39 46 NA NA rs300291 0,941 1,000 1 1,000 C29 Rf1821 1 c.1956+7C>T g.2705812C>T S5 P-ORF intron 14 40 NA NA rs145755468 5,24E-04 3,93E-03 0 7,41E-04 C30 Rf1711 1 F c.2063+20G>T g.2706488G>T S5 P-ORF intron 15 28 -6 20 rs368528253 C31 Rf2520 1 c.3425+37C>T g.2738580C>T S5 P-ORF intron 26 47 NA NA rs8094260 0,276 0,285 0,504 0,466 C32 Rf1795 1 c.4966+31delA g.2770137delA S5 P-ORF intron 39 Normal NA NA rs547466679 8,15E-04 5,85E-03 0 1,13E-03 C33 34 1 c.5476+10A>G g.2777923A>G S5 P-ORF intron 43 Normal Normal NA rs3213926 0,259 0,302 0,566 0,419 C34 Rf1838 1 c.5878+8T>G g.2796113T>G S5 P-ORF intron 46 52 NA NA rs144115061 3,83E-04 1,60E-03 0 0 C35 Rf2704 3 M c.639-9T>G g.2688383T>G S3 D-ORF intron 5 Normal Normal NA C36 Rf799 1 c.2147-7C>T g.2707798C>T S3 P-ORF intron 16 Normal Normal NA rs113524119 9,10E-04 6,96E-03 0 5,93E-04 C37 Rf701 4 F c.3802-16T>G g.2747504T>C S3 P-ORF intron 29 Normal Normal NA rs79829175 6,48E-02 1,78E-04 0 6,37E-05 C38 Rf2384 1 M c.262+25G>C g.2666256G>C S5 P-ORF intron 2 Normal Normal NA rs634246 0,549 0,071 5,79E-04 0,0395 C39 Rf2384 1 M c.262+48T>C g.2666279T>C S5 P-ORF intron 2 Normal Normal NA rs531379 0,516 0,059 0,001 0,0362 C40 Rf2384 1 M c.424+33T>C g.2667063T>C S5 P-ORF intron 3 Normal Normal NA rs648105 0,532 0,059 0 0,0423 C41 Rf976 1 F c.873+33delT g.2688776delT S5 P-ORF intron 7 Normal Normal NA C42 Rf2384 1 M c.2603+25T>G g.2722686T>G S5 P-ORF intron 20 Normal Normal NA rs73365837 0,162 7,22E-04 1,16E-04 1,87E-04 C43 Rf2384 1 M c.3634-35C>T g.2743724C>T S5 P-ORF intron 28 Normal Normal NA rs59023685 0,172 8,01E-04 1,36E-04 1,89E-04 C44 Rf2384 1 M c.5052+33dupT g.2771643A>AT S5 P-ORF intron 40 Normal Normal NA rs33935864 0,252 0,280 0,516 0,440 C45 Rf1565 1 M c.159C>T; c.2147-7C>T g.2656233C>T; g.2707798C>T Y53Y SYN P-ORF exon 1 70 NA NA rs1206890396 C46 4 1 c.174G>C g.2656248G>C A58A SYN P-ORF exon 1 Normal Normal NA rs2430853 0,852 0,551 0,343 0,637 C47 Rf1858 1 F c.1419A>G + c.1648-31A>G g.2700613A>G; g.2703659A>G E473E SYN P-ORF exon 11 Normal Normal NA rs117771893 4,60E-04 4,44E-03 0 2,43E-03 C48 Rf2520 1 c.1608A>G g.2700877A>G K536K SYN P-ORF exon 12 47 NA NA rs72862973 0 2,11E-04 0 0 C49 Rf1785 1 M c.1689G>A g.2703731G>A L563L SYN P-ORF exon 13 46 NA NA rs78073508 2,02E-02 3,00E-05 0 6,06E-05 C50 12 1 c.1851A>G g.2705700A>G T617T SYN P-ORF exon 14 Normal Normal NA rs635132 0,335 0,723 0,674 0,649 C51 Rf955 3 M c.2433T>G g.2718407T>G S811S SYN P-ORF exon 19 58 11 44 rs760899591 0 3,00E-05 0 0 C52 Rf1824 1 c.2529C>A g.2722587C>A I843I SYN P-ORF exon 20 51 NA NA rs761184318 0 0 0 1,21E-04 C53 Rf1658 1 M c.2925T>A g.2729284T>A A975A SYN P-ORF exon 24 Normal NA NA rs763236583 0 8,47E-05 0 0 C54 24 1 c.3528A>G g.2740714A>G T1176T SYN P-ORF exon 28 Normal Normal NA rs12327477 0,270 0,280 0,504 0,450 C55 Rf1830 1 c.3537C>T g.2740723C>T Y1179Y SYN P-ORF exon 28 46 NA NA rs372029124 1,02E-04 3,00E-05 0 9,09E-04 C56 27 1 c.4137A>G g.2750477A>G A1379A SYN P-ORF exon 32 Normal Normal NA rs2304859 0,297 0,325 0,533 0,471 C57 Rf1158 1 M c.4281T>C g.2751391T>C N1427N SYN P-ORF exon 33 57 NA NA rs757680634 0 4,62E-05 0 0 C58 29 1 c.4629C>T g.2763697C>T G1543G SYN P-ORF exon 37 Normal Normal NA rs483547 0,301 0,450 0,174 0,198

X1 Rf653 1 F c.1939G>A g.2705788G>A E647K M P-ORF exon 14 28 -16 16 possibly_damaging(0.715) Deleterious(0.01) C15 5 29 1 X2 Rf668 1 M c.4G>C g.2656078G>C A2P M P-ORF exon 1 18 -12 12 probably_damaging(0.981) Tolerated(0.11) C0 238 2 1 X2 Rf668 3 F c.4G>C g.2656078G>C A2P M P-ORF exon 1 24 -15 14 probably_damaging(0.981) Tolerated(0.11) C0 238 2 1 Supplementary table 2 Table summarizing the variant analysis in 187 unrelated FSHD2 families (F1-F187), 41 BAMS (B1-B41) families, 58 non-pathogenic variants (C1-C58) and 2 for which the pathogenicity is unclear (X1-X2), with proband in white and family members carrying the variant in grey (column 1). For each individual the family, personal number (nr) and gender (if known) is shown (columns 2, 3 and 4). For each variant, we provide cDNA (based on accession number NM_015295.2), genomic (based on hg19, GRCh37.p5) and protein (NP_056110.2) information (columns 5, 6 and 7). Column 8 describes the variant type (M=missense, D=insertion/deletion, N=nonsense, S3/S5 splice site variant at the 3’ or 5’ and SYN=synonymous). The variants are sorted by type and by position. ORF-disrupting (D-ORF) and ORF-preserving (P-ORF) consequence of the variant and the SMCHD1 exon number is shown in columns 9 and 10. The D4Z4 methylations values; FseI, delta 1 and delta2 for the individual are shown in columns 11,12 and 13. Values that were not determined or could not be calculated were indicated with NA. And for control individuals, variants that were analyzed in multiple individuals showing normal methylation values were marked normal. The dbSNP number and the frequency of the variant in the EXAC database (AFR=African, EUR=European; EAS=East Asian and SAS=South Asian populations) in columns 14-18). For missense variants we evaluated the pathogenic effect by the following prediction algorithms: PolyPhen (from Variant Effect Predictor in Ensemble; column 19), SIFT (from Alamut, column 20) and Align GVGD Class (columns 21, 22 and 23). Each program has a different pathogenicity score. A pathogenic prediction within one of the programs received a score of 1 (marked grey) and a benign prediction a score of 0 (marked white). The total prediction score for all 3 algorithms is shown in column 24, where we highlighted the variants in grey that obtained a false negative prediction (D4Z4 hypomethylation, but total prediction score <2), or a false positive prediction (normal D4Z4 methylation, but total prediction score >1). The last column shows the publication, in which the variant was first described.

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Mutation other published new variants spectrum 20153 variants current study total variants n % n % n % n % Indel 8 15,7% 11 20,8% 16 19,3% 35 18,7% Missense 13 25,5% 20 37,7% 21 25,3% 54 28,9% Nonsense 5 9,8% 5 9,4% 18 21,7% 28 15,0% Splice site 25 49,0% 17 32,1% 28 33,7% 70 37,4% Total 51 53 83 187

Supplementary table 3 SMCHD1 variant type for all variants published in our previous study (n=51)3, in 12 other publications (n=53, Supplementary table 1)5-15, and new variants (n=83) in the current study.

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ATPase Remaining Distribution domain % protein % P value Total Exons 3-12 1-2 / 13-48 Amino acids 445 22,2% 1560 77,8% NA 2005 Indels1 10 28,6% 25 71,4% 0,49 35 Missense 27 50,0% 27 50,0% 3,61E-06 54 Nonsense 7 25,0% 21 75,0% 0,90 28 Splicing (S3) 1 5,9% 17 94,4% 0.162 182 Splicing (S5) 8 15,4% 44 84,6% 0,32 52 351 1 Complete gene deletions were not included 2 Chi-squared approximation may be incorrect

Supplementary table 4 Distribution of the 187 FSHD2-related SMCHD1 variants in the C-terminal extended ATPase domain and in the rest of the protein. C-terminal extended ATPase domain is based on Gordon et al. 2017.16 In contrast to the other variant types, we observe a significant enrichment (P value 1.59E-05) of missense variants in the extended ATPase domain compared with the size (445/2005 amino acids) of this region.

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Proband ID Sex cDNA Protein Exon Inheritance PMID publication K1 F c.320T>C L107P 3 NA 2806790917 D1 M c.386T>A M129K 3 NA 2806790917 New F c.386T>G M129R 3 NA new 11 F c.400G>T A134S 3 de novo 2806791116 12 F c.400G>T A134S 3 de novo 2806791116 M1 and 2* F c.403A>T S135C 3 de novo 2806790917/2806791116 AF1 and 4* F c.403A>T S135C 3 de novo 2806790917/2806791116 I1 M c.404 G>A S135N 3 de novo 2806790917 R1 F c.404G>A S135N 3 Probably familial 2806790917 3 M c.404G>A S135N 3 de novo 2806791116 AK1 M c.404G>T S135I 3 de novo 2806790917 1 M c.407A>G E136G 3 de novo 2806791116 T1 M c.408A>C E136D 3 Paternal 2806790917 AG1 F c.410 G>A G137E 3 NA 2806790917 A1 F c.415A>C N139H 3 de novo 2806790917 Y1 F c.415A>C N139H 3 NA 2806790917 C1 M c.423G>C L141F 3 NA 2806790917 E1 M c.423G>C L141F 3 NA 2806790917 S1 F c.423G>C L141F 3 NA 2806790917 V1 M c.423G>T L141F 3 de novo 2806790917 AB1 M c.511T>G F171V 5 Probably familial 2806790917 AA1 M c.725C>G A242G 6 de novo 2806790917 10 F c.1025G>C W342S 8 de novo 2806791116 O1 F c.1034A>G Q345R 8 Maternal 2806790917 F1 M c.1043A>G H348R 9 NA 2806790917 L1 and 13* F c.1043A>G H348R 9 NA 2806790917/2806791116 N1 and 5* M c.1043A>G H348R 9 de novo 2806790917/2806791116 X1 F c.1043A>G H348R 9 de novo 2806790917 Z1 M c.1043A>G H348R 9 NA 2806790917 AC1 M c.1043A>G H348R 9 de novo 2806790917 AE1 M c.1043A>G H348R 9 de novo 2806790917 14 F c.1043A>G H348R 9 NA 2806791116 AH1 F c.1199A>T Q400L 10 Paternal 2806790917 P1 and 6* M c.1259A>T D420V 10 de novo 2806790917/2806791116 9 M c.1259A>T D420V 10 de novo 2806791116 W1 M c.1417G>C E473Q 11 NA 2806790917 8 F c.1552A>G K518E 12 NA 2806791116 J1 M c.1568C>A T523K 12 NA 2806790917 U1 F c.1568C>A T523K 12 NA 2806790917 B1 M c.1571A>G N524S 12 NA 2806790917 AJ1 and 7* M c.1655G>A R552Q 13 de novo 2806790917/2806791116

Supplementary table 5 Overview of SMCHD1 variants identified in 41 BAMS families from previous publications.16, 17 The 1st and 2nd columns show the reference ID and gender of the proband. The 3rd, 4th and 5th columns shows the position of the variant in the cDNA, the protein and the exon and whether the variants occurred de novo, or not. The PMID of the publication describing the family is shown in the last column.

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Supplementary Figures

Normal (8-100U) DUX4

D4Z4 repeat with D4Z4 repeat array D4Z4 unit closed chromatin D4Z4 repeat with FSHD1 FSHD2 open chromatin 1-10U D4Z4 repeat 8-20U D4Z4 repeat Wildtype SMCHD1 + SMCHD1 variant Mutant SMCHD1

DUX4 DUX4

Supplementary figure 1 FSHD is caused by mis-expression of the transcription factor DUX4 in skeletal muscle, where it is normally repressed. A complete copy of the DUX4 retrogene is embedded in the most distal unit of the D4Z4 macrosatellite repeat on 4 and the region immediately distal to the repeat. In control individuals the repeat varies between 8-100 units, In most FSHD cases, the disease is caused by a D4Z4 repeat contraction to a size of 1-10 units (FSHD1). The less common form FSHD2 is caused by heterozygous variants in the chromatin modifier SMCHD1 in combination with a D4Z4 repeat of 8-20 units, also resulting in DUX4 expression in skeletal muscle. Individual D4Z4 units are depicted as open and filled (representing open and closed chromatin structure) triangles, DUX4 protein expression is indicated with diamonds. Wildtype and mutant SMCHD1 protein are indicated with a closed and open symbol, respectively.

8

80

20 50

60

0

40 25

FseI

delta2 delta1 -20

20

0

-40

0

control FSHD2 control FSHD2 control FSHD2

Supplementary figure 2 Threshold methylation values for control individuals (n=249) and for unrelated FSHD2 patients in this study (n=89). The red vertical line with dots indicates the average methylation and 1 SD, while the red box indicates the control and FSHD2 threshold for the different methylation values (1.5 or 2 SD). The FseI methylation value shows the highest variability due to the contribution of the D4Z4 repeat size. The average methylation in controls is 46.8% with a SD of 14.1% and the threshold for FSHD2 is defined at 25% (1.5 SD below the average). The delta1 value has an average value in controls of 0% with an SD of 10.0% and the threshold for FSHD2 is defined at -20.0% (2 SD below the average). We also define a delta1 grey zone between -15.0% to -20.0% where the milder methylation defect might occur due to variants in unknown epigenetic modifiers. The delta2 value is only valid for SMCHD1 mutation carriers. The average is -0.7% and the standard deviation is 4.7%.

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* *

Supplementary figure 3 Delta2 methylation values for carriers of the same SMCHD1 variant in comparison to the distribution of all carriers of a disrupting ORF (D-ORF, left) or preserving ORF (P-ORF, right) variant. For most variants the methylation level of different carriers is comparable. Also different intron 25 variants which probably all result in the skip of exon 25 have a comparable delta2 methylation value (P-ORF, right). Individuals with exceptionally high D4Z4 methylation levels discussed in the text are marked with an asterisk.

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D-ORF P-ORF ATPase domain non-ATPase domain Missense P-ORF variants

Supplementary figure 4 Violin plot with delta2 methylation values for unrelated carriers of an D-ORF or P-ORF SMCHD1 variant (left) and for ATPase domain P-ORF missense variants and the other P-ORF variants (right). The lines indicate the average, the 25th and 75th percentiles.

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Rf668

WT/c.4G>C WT/WT [6U(95%);16U(5%)] 4A161 59U 4A161 18U 4B163 45U 4B168 FseI: 18% FseI: NA delta1: -12% delta1: NA delta2: +12% delta2: NA

WT/c.4G>C WT/WT 16U 4A161 16U 4A161 59U 4A161 45U 4B168 FseI: 24% FseI: 24% delta1: -15% delta1: -13% delta2: +14% delta2: +15%

Supplementary figure 5 Pedigree of family Rf668. Father and both daughters have an FseI methylation below the FSHD2 threshold and low, but normal, delta1 scores. The father and oldest daughter are heterozygous for the SMCHD1 variant A2P, while the youngest daughter does not carry the variant. The first column shows information on the SMCHD1 variant, the 2nd and 3rd columns show repeat size and haplotype of both D4Z4 alleles at chromosome 4. The other columns show information on the FseI, delta1 and delta2 methylation values.

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Supplementary figure 6

Comparison of four GHKL ATPase dimers and their downstream ,or C-terminal extended, domains. (A) Computational model of SMCHD1, (B) E. coli MutL, (C) MORC2 and (D) Mycobacterium tuberculosis GyrB. In each panel, the two ATPase domains are colored in green and cyan, with a helix and loop at the dimer interface marked in dark blue. In SMCHD1, several BAMS mutations localize to this loop. Downstream domains (M-domain, transducer domain) are colored in grey. The ATPase domains show strong structural conservation, while the downstream domains show a much greater structural diversity.

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14 reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy. Hum Mutat 2017;38:1432-41. 13. Sacconi S, Lemmers RJ, Balog J, van der Vliet PJ, Lahaut P, van Nieuwenhuizen MP, Straasheijm KR, Debipersad RD, Vos-Versteeg M, Salviati L, Casarin A, Pegoraro E, Tawil R, Bakker E, Tapscott SJ, Desnuelle C, van der Maarel SM. The FSHD2 gene SMCHD1 is a modifier of disease severity in families affected by FSHD1. Am J Hum Genet 2013;93:744-51. 14. van den Boogaard ML, Lemmers RJ, Camano P, van der Vliet PJ, Voermans N, van Engelen BG, Lopez de MA, Tapscott SJ, van der Stoep N, Tawil R, van der Maarel SM. Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2. Eur J Hum Genet 2016;24:78-85. 15. Winston J, Duerden L, Mort M, Frayling IM, Rogers MT, Upadhyaya M. Identification of two novel SMCHD1 sequence variants in families with FSHD-like muscular dystrophy. Eur J Hum Genet 2015;23:67-71. 16. Gordon CT, Xue S, Yigit G, Filali H, Chen K, Rosin N, Yoshiura KI, Oufadem M, Beck TJ, McGowan R, Magee AC, Altmuller J, Dion C, Thiele H, Gurzau AD, Nurnberg P, Meschede D, Muhlbauer W, Okamoto N, Varghese V, Irving R, Sigaudy S, Williams D, Ahmed SF, Bonnard C, Kong MK, Ratbi I, Fejjal N, Fikri M, Elalaoui SC, Reigstad H, Bole-Feysot C, Nitschke P, Ragge N, Levy N, Tuncbilek G, Teo AS, Cunningham ML, Sefiani A, Kayserili H, Murphy JM, Chatdokmaiprai C, Hillmer AM, Wattanasirichaigoon D, Lyonnet S, Magdinier F, Javed A, Blewitt ME, Amiel J, Wollnik B, Reversade B. De novo mutations in SMCHD1 cause Bosma arhinia microphthalmia syndrome and abrogate nasal development. Nat Genet 2017;49:249-55. 17. Shaw ND, Brand H, Kupchinsky ZA, Bengani H, Plummer L, Jones TI, Erdin S, Williamson KA, Rainger J, Stortchevoi A, Samocha K, Currall BB, Dunican DS, Collins RL, Willer JR, Lek A, Lek M, Nassan M, Pereira S, Kammin T, Lucente D, Silva A, Seabra CM, Chiang C, An Y, Ansari M, Rainger JK, Joss S, Smith JC, Lippincott MF, Singh SS, Patel N, Jing JW, Law JR, Ferraro N, Verloes A, Rauch A, Steindl K, Zweier M, Scheer I, Sato D, Okamoto N, Jacobsen C, Tryggestad J, Chernausek S, Schimmenti LA, Brasseur B, Cesaretti C, Garcia-Ortiz JE, Buitrago TP, Silva OP, Hoffman JD, Muhlbauer W, Ruprecht KW, Loeys BL, Shino M, Kaindl AM, Cho CH, Morton CC, Meehan RR, van H, V, Liao EC, Balasubramanian R, Hall JE, Seminara SB, Macarthur D, Moore SA, Yoshiura KI, Gusella JF, Marsh JA, Graham JM, Jr., Lin AE, Katsanis N, Jones PL, Crowley WF, Jr., Davis EE, Fitzpatrick DR, Talkowski ME. SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome. Nat Genet 2017;49:238-48.

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